Abstract Pulmonary arterial hypertension (PAH) is a disease of pulmonary vasculature with a high mortality rate of up to 45% three years after diagnosis. PAH is often associated with the loss of endothelial vasodilation, which has long been thought to be a major contributor to development of PAH. Endothelial dysfunction in PAH results in reduced release of the predominant vasodilatory molecule in pulmonary vasculature? nitric oxide (NO); but the underlying pathological mechanisms remain unknown. A detailed mechanistic understanding of the pathways that regulate endothelial NO synthase (eNOS) activity in pulmonary vasculature is necessary for deciphering the mechanisms that cause endothelial dysfunction in PAH. In this application we focus on local, unitary Ca2+ influx events through endothelial TRPV4 (transient receptor potential vanilloid 4) channels?TRPV4 sparklets? that regulate eNOS activity and NO release in the native pulmonary artery endothelium. Our preliminary results reveal that TRPV4 is a major Ca2+ influx pathway in pulmonary endothelium that causes vasodilation through eNOS activation. Moreover, endothelial TRPV4 channels serve as a control mechanism that integrates information from different physiological stimuli, including adenosine triphosphate (ATP), flow/shear stress, and smooth muscle ?1 adrenergic stimulation, via distinct signaling mechanisms. The TRPV4-induced vasodilations and NO release are impaired in chronic hypoxia-induced PAH. We hypothesize that impaired TRPV4 Ca2+ signaling is responsible for the loss of endothelial vasodilations in PAH. In Specific Aim 1, we will determine the role of endothelial TRPV4 channels as a key control mechanism that integrates information from different physiological stimuli via distinct signaling pathways to cause vasodilations in mouse and human pulmonary arteries. We will also confirm the physiological roles of endothelial TRPV4 channels in endothelium- specific TRPV4-/- mice. In Specific Aim 2, we will define the TRPV4-eNOS linkage that causes vasodilation in response to physiological stimuli. We will also determine how NO itself regulates TRPV4 channel activity through endothelial guanylyl cyclase-protein kinase G (PKG) mechanism. In Specific Aim 3, we will delineate the TRPV4-eNOS dysfunction in mouse models of PAH, and test the hypothesis that endothelial PKG upregulation is responsible for impaired function of TRPV4-eNOS axis in PAH. These studies will result in novel discoveries including first evidence of eNOS regulation by localized Ca2+ signals and its impairment in PAH. Results from these studies will provide much needed novel therapeutic targets for treating PAH.